These investigations will focus on microRNA-mediated mRNA degradation in mammalian cells, with the goal of understanding the mechanism by which microRNAs expedite deadenylation as an early step in the degradation of their mRNA targets and the contribution of mRNA deadenylation and decay to gene regulation by microRNAs. Using cultured mammalian cells as model systems and molecular biological methods as analytical tools, we will investigate the proteins that contribute to microRNA-directed deadenylation, the manner in which they associate with one another, and the importance of those interactions for miRNA function. In addition, we will examine the impact of mRNA context on the degree to which microRNAs influence mRNA stability. Finally, we will explore the mechanism by which microRNAs identify their target sites in mRNA. The results of these studies should enhance our knowledge of a widespread mechanism of eukaryotic gene regulation that presently is poorly understood. This knowledge should ultimately be of value for understanding the etiology of diseases thought in some instances to be related to defects in microRNA function and for improving the efficacy of RNA interference as a medical therapy. Lay language statement: There is growing awareness that the hundreds of distinct microRNAs in human cells play key roles in gene regulation. Additional evidence suggests that microRNAs are important for viral infection and that defects in microRNA function can contribute to cancer and congenital abnormalities. The proposed research should eventually be of value for understanding the etiology of diseases related to microRNA function or dysfunction and for improving the efficacy of medical therapies based on small RNA molecules.